Construction and expression of Ca2+/calmodulin-dependent protein kinase Ⅱ plasmid and identification of Cav1.2 channel binding
10.12007/j.issn.0258-4646.2025.01.001
- VernacularTitle:钙/钙调蛋白依赖性蛋白激酶Ⅱ质粒的构建和表达及其与Cav1.2通道结合的鉴定
- Author:
Hongmei WANG
1
;
Xianghui WANG
;
Wenzhu ZHANG
;
Rui HE
;
Tianzuo LIAO
;
Qinghua GAO
;
Liying HAO
Author Information
1. 中国医科大学药学院药物毒理学教研室,沈阳 110122;赤峰市医院药剂科,内蒙古赤峰 024000
- Publication Type:Journal Article
- Keywords:
Ca2+/calmodulin-dependent protein kinase Ⅱ;
plasmid construction;
extraction;
purification;
pull-down
- From:
Journal of China Medical University
2025;54(1):1-4,11
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.
